EPA-660/3-73-018
January 1974
Ecological Research Series
Effects of Crude Oil and
Some of Its Components on
Young Coho and Sockeye Salmon
Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C. 20460
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RESEARCH REPORTING SERIES
Research reports of the office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
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was consciously planned to foster technology
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1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL
RESEARCH series. This series describes research
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assessed for their long- and short-term
influences. Investigations include formation,
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EPA-660/3-73-018
January 1974
EFFECTS OF CRUDE OIL AND SOME OF ITS COMPONENTS
ON YOUNG COHO AND SOCKEYE SALMON
by
James E. Morrow
Project R801039 (formerly 16100FWQ)
Program Element 1BA022
Project Officer
Dr. Ronald C. Gordon
Arctic Environmental Research Laboratory
Environmental Protection Agency
Fairbanks, Alaska 99701
Prepared for
Office of Research and Development
U. S. Environmental Protection Agency
Washington, D. C. 20460
For rale by the Superintendent ot Document!. U.S. Government Printing Office, Washington, D.C. 20402 • Price 85 cents
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ABSTRACT
Young coho and sockeye salmon, acclimated to 30 °/oo salinity, were
exposed in various ways to different amounts of crude oil from the
Prudhoe Bay field. Oil poured on the surface of the water in 95 liter
(25 gallon) aquaria produced significant mortalities when the oil con-
centration was 500 ppm or greater. Fish dipped into a crude oil film,
or with a drop of oil placed directly on each gill, showed no signifi-
cant mortalities. The same was true of fish force-fed crude oil at
1 g per 100 g body weight. Oil that had been exposed to air for 30
days produced no significant mortalities.
Among oil components tested for toxicity on coho salmon, aliphatic
compounds were not lethal. Mono-cyclic aromatics were generally toxic,
the degree of toxicity increasing with the degree of unsaturation.
It is suggested that the toxicity of these substances is brought about
through alteration of cell membrane permeability, especially in the
gills. This results in a rapid increase of mono-valent ions in the
blood and probably also interferes with CO.-HCO. regulation.
This report submitted in fulfillment of Project R80103& (formerly
16100FWQ) by the Department of Biological Sciences, University of Alaska,
Fairbanks, Alaska, under the sponsorship of the Environmental Protec-
tion Agency. Work was completed in May 1973.
11
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CONTENTS
Abstract ii
List of Tables iv
Acknowledgments vi
Sections
I Conclusions 1
II Recommendations 2
III Int r oduct i on 3
IV Materials and Methods 5
V Results 9
VI Discussion 34
VII References 37
111
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TABLES
No. Page
1 Changes in serum chloride of young sockeye salmon
exposed to various amounts of crude oil at 8° C 10
2 72 hour mortalities in young coho salmon exposed to
crude oil in experiment I-C-1 12
3 96 hour mortalities in young coho salmon exposed to
crude oil in experiment I-C-1 13
4 Mortalities observed in young coho salmon exposed to
weathered crude oil in experiment I-C-2 14
5 96 hour mortalities in young coho salmon exposed to
crude oil at 8° C in experiment I-C-3 15
6 96 hour mortalities in young sockeye salmon exposed to
different amounts of crude oil at 8° C in experiment
I-S-1 16
7 96 hour mortalities in young sockeye salmon exposed to
different amounts of crude oil at 3°-5° C in experiment
I-S-2 17
8 96 hour mortalities in young sockeye salmon exposed to
different amounts of crude oil at 13° C in experiment
I-S-3 18
9 96 hour mortalities in young coho salmon exposed to
mixed or unmixed crude oil at 8° C in experiment II-C-1 19
10 Mortalities observed in experiments III-C-1, IV-C-1,
III-S-1, and IV-S-1 21
11 Mortalities produced with young coho salmon and 50 ppm
1,3 cyclohexadiene at 8° C 23
12 Mortalities produced with young coho salmon and
benzene at 8° C 24
13 Mortalities produced with young coho salmon and
ethylbenzene at 8° C 25
14 Mortalities produced with young coho salmon and
xylene at 8° C 26
IV
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TABLES
No. Page
15 Mortalities produced with young coho salmon and
toluene at 8* C 27
16 Changes in blood sodium and chloride ion of young
coho salmon after exposure to 100 ppm cyclohexene
at 8° C 29
17 Changes in blood sodium and potassium of young coho
salmon after exposure to 15 ppm benzene at 8° C 30
18 Changes in blood sodium and potassium of young coho
salmon after exposure to 30 ppm xylene at 8° C 31
19 Changes in blood sodium and chloride of young coho
salmon after exposure to 30 ppm xylene at 8° C 32
20 Changes in blood sodium and potassium of young coho
salmon after exposure to 30 ppm toluene at 8° C 33
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ACKNOWLEDGMENTS
I thank Mr. John Sweet, Atlantic Richfield Oil Company, for providing
the crude oil used in Phase I of this study; the Alaska Department
of Fish and Game, particularly Mr. Joe Wallis and his staff at the
Fire Lake Hatchery, for the fish used as experimental animals; the
Arctic Environmental Research Laboratory, Environmental Protection
Agency, Fairbanks, Alaska, for the use of space and facilities; and
the staff of that laboratory, especially Dr. Ronald C. Gordon, for
their cooperation and encouragement.
vi
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SECTION I
CONCLUSIONS
Under laboratory conditions which attempted to duplicate a natural
environment, as far as this could be done within the confines of 95
liter (25 gallon) aquaria, young coho and sockeye salm'on are vulner-
able to crude oil slicks when the amount of oil present is in the
equivalent of 500 ppm or greater. Fish so exposed exhibit a typical
behavior pattern, and the majority die within 96 hours. At lower con-
centrations of oil, the behavior pattern is less marked and mortalities
are lower.
Aliphatic compounds are relatively innocuous, whether they be saturated
or mono-unsaturated. By contrast, monocyclic aromatic compounds are
generally toxic, and the degree of toxicity increases with increasing
unsaturation.
Toxicity of unsaturated aromatic compounds probably results from alter-
ation of cell membrane permeability, especially in the gills, by disso-
lution of fatty substances from them. This, in turn, destroys the
ability to regulate salt balance. The monovalent ions sodium, potassium,
anc chloride increase rapidly but temporarily in the blood, and pro-
bably in the tissues as well. The observed symptoms are muscle hyper-
tension, hyperactivity, loss of equilibrium, and death. These symp-
toms are consistent with the thesis that ionic imbalance in blood and
tissues, together with loss of control of C02-HCO~ balance, are the
active mechanisms.
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SECTION II
RECOMMENDATIONS
The theories presented in this report should be tested, utilizing more
sophisticated instrumentation and techniques and larger animals.
We suggest that the matter of altered cell membrane permeability could
be determined by perfusion techniques on excised gills. Swim bladder
gases should be analyzed to determine whether any change in composition
appears after exposure to aromatic substances. Blood pH and C0~ should
be measured at the same time, with the blood taken from the aorta close
to the gills. This should determine whether the COj-HCOl balance is
actually disturbed.
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SECTION III
INTRODUCTION
GENERAL
The discovery of oil on the North Slope of Alaska and the construction
of the proposed trans-Alaska pipeline from Prudhoe Bay to Valdez present
the threat of oil pollution in one of the major fisheries areas of
Alaska. Until recently, Alaskan fisheries have been singularly free of
this sort of pollution, but as the possibility of transporting vast
quantities of oil from the port of Valdez increases, so also does the
probability of oil pollution in the area. Since the fisheries are one
of the most valuable industries in the state and will undoubtedly con-
tinue for a long time to occupy this position in the economy, it is
necessary to investigate the effects of oil on the fisheries in order
to be able to predict, ameliorate, and, hopefully, avoid altogether
damage to this major source of income and employment. The present
project was designed to contribute information that might be applied
towards this goal, by determining the toxicity levels of crude oil and
some of its components and also by attempting to determine the mecha-
nisms of this toxicity.
The area of greatest interest from the standpoint of the relationship
of oil pollution to the fisheries is Prince William Sound and its imme-
diate environs. For this reason, laboratory conditions were designed
to duplicate, as nearly as possible, the conditions in that body of
water. All experiments were conducted in artificial sea water of approx-
imately 30 °/oo salinity and at temperatures between 3° and 13° C.
These represent average salinity and minimum and maximum temperatures
to be expected in the area (7). It was not practical to try to trans-
port the needed quantities of natural sea water from the coast to Fair-
banks, nor were storage facilities for sea water available. Artificial
sea water offered the further advantage of being uncontaminated by plank-
ton, whose metabolic activities would have added unknown and probably
unmeasurable factors to the experiments.
The choice of experimental animals was dictated by a number of factors.
Pragmatically, those species most valuable to the fisheries should be
the first choice, and here the several species of Pacific salmon (genus
Oncorhynchus) lead all the rest. Among the salmon, the chum (0_. keta)
and the sockeye (0_. nerka) offered a number of advantages. In parti-
cular, the presumed availability of sockeye from the hatcheries of the
Alaska Department of Fish and Game made this species a logical choice.
Chums are not cultured in Alaska. Unfortunately, when the project fi-
nally got under way, sockeye were not available. We turned to the coho
salmon C£- kisutch) which are rather more difficult to acclimate to salt
water, but which were readily obtainable at the time. Later, when
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sockeye were once again available, some experiments were run with this
species.
The study was established in two phases. The first involved exposure
of experimental fish to various amounts of crude oil from the Prudhoe
Bay field in northern Alaska, while in the second phase the fish were
exposed to various individual components of crude oil.
OBJECTIVES
Phase I was designed to determine the degree to which crude oil would
affect fishes and to show the mechanisms by which the effects were
brought about. Phase II attempted to do the same thing for several
classes of hydrocarbons which are normally found in crude oil, thus
hopefully identifying the particular groups of compounds responsible
for the effects observed in Phase I.
As a result of some suggestive data accumulated in the first part of
this study, we had planned to analyze swim bladder gases during the
second phase of investigations. Unfortunately, the fish that were
available during the second stage were much too small to allow this.
Indeed, most of the experimental fish of the second phase were so small
that blood analyses had to be curtailed. We could not obtain enough
blood from .single specimens to run complete analyses.
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SECTION IV
MATERIALS AND METHODS
GENERAL
Young coho (Oncorhynchus kisutch) and sockeye (0_. nerka) salmon were
obtained from the Fire Lake Hatchery of the Alaska Department of Fish
and Game. These fish were derived from local Alaskan stocks, the coho
from Ship Creek, near Anchorage, and the sockeye from Bear Creek, near
Seward. At the hatchery, the fish were placed in plastic bags of water,
the bags sealed and put in styrofoam shipping boxes. They were then
trucked to the Anchorage airport (about 20 miles [32 km]) and sent by
air freight to Fairbanks. Except for one shipment, mortalities due ta
this handling were always less than 1%. The exceptional shipment had
apparently been left on a loading dock in full sunshine for about six
hours. More than half the fish in that shipment were dead on arrival
at Fairbanks.
At the laboratory, the fish were put into two large circular tanks,
each containing approximately 1135 liters of conditioned fresh water.
The water in these tanks was kept at 8° C and circulated by a Min-0-
KooL chiller-circulator. Filtration was done by pumping the water
through a home-made fiberglass-charcoal filter with a small submersible
pump. Waste material was removed once or twice a day. Fish in the
stock tanks were fed five days a week with Oregon fish pellets.
After the fish had become acclimated to the stock tanks (usually two to
four days, judged by their behavior in the tanks), Instant Ocean brand
artificial sea salts were added over a period of about a week to bring
the salinity up to approximately 30 °/oo. The fish were allowed to
acclimate to the salt water for about another week before being used in
experiments. Time of acclimation varied between 3 and 10 days, depen-
ding on size and species. Sockeye salmon adapted much more readily
than did coho, and larger individuals, especially those beginning to
"smolt up," adapted more easily than did smaller ones.
Experiments were conducted in Instant Ocean brand aquaria of 25 gallon
(95 liter) dapacity, each containing 73 liters of conditioned salt water
from the stock tanks. The gravel-like filtration material that is a
standard part of these aquaria was not used in these experiments, as it
would have been impossible to clean after being fouled with the experi-
mental substances. The squaria were fully aerated and each was covered
with a sheet of glass. Details of experimental procedures are given
below.
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PHASE I
Five series of experiments were done on coho salmon. With one exception
(the final experiment of this phase), analyses were done after 96 hours.
In Series I, measured amounts of crude oil were poured on the surface
of the water. Behavior of the fish was observed, mortalities recorded,
blood parameters (Na+, K+, C1-, C02, 02, pH, serum proteins) analyzed,
tissue samples (gill stomach, pyloric caeca, intestine, liver, fatty
tissue, kidney, spleen) removed and fixed in Bouin's fluid. Experi-
ments and controls were replicated at 3°, 8°, and 13° C. Environmental
parameters monitored were 02, C02, pH, H2S, NHs, salinity, copper,
zinc, iron, and lead. Dead fish were removed daily.
For the blood parameters, serum Na+ and whole blood K+ were determined
with a Coleman model 6-20 flame spectrophotometer. Serum Cl~ was ti-
trated with an Oxford microtitrometer, following the Oxford modification
of the method of Schales and Schales (6). C02, 02, and pH were meas-
ured with a Corning model 16 blood pH/gas analyzer. Serum proteins
were examined by disc gel electrophoresis with a Canalco model 1200
electrophoresis apparatus with scanner and printer.
Among the environmental parameters, salinity was determined with a
Beckman model RS7-B salinometer; pH was read on a Coleman model 38A
pH meter; oxygen was measured by the modified Winkler method arid C02
was calculated from pH values of acidified samples (8). H2S, NHs, and
heavy metals were determined by personnel of the Arctic Environmental
Research Laboratory, Environmental Protection Agency.
Series II was the same as Series I, except that the oil was physically
mixed in each tank by means of a stream of water (approximately 19
liters/minute) for five minutes at the beginning of each experiment.
In both Series I and II, the fish to be analyzed were anesthetized with
MS 222 (Tricaine methane sulfonate). After trying narious methods, we
concluded that the most satisfactory way of obtaining blood was to cut
.off the animal's tail and take blood from the caudal aorta. This
method was used throughout the study.
In Series III,, fish were anesthetized with MS 222, then dipped into a
film of crude oil to simulate leaping or swimming through an oil spill.
The fish-were then placed in tanks of clean, aerated water, and excess
oil carried into the tanks on the bodies of the fish was skimmed off the
surface of the water. Mortalities were recorded and specimens were
sacrificed at intervals for study of gill tissues. Controls were
treated similarly, except that they were dipped into clean water in-
stead of oil.
Experimental procedure of Series IV consisted of placing one drop of
crude oil directly on the first gill on each side of an anesthetized
6
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fish. The fish were then placed in tanks of clean water. Data were
recorded as for Series III.
In Series V, crude oil in amount of Ig/lOOg body weight was forced
gently into the stomach of each anesthetized fish from a syringe,
The fish were put into tanks of clean salt water and observed, Experi-
mental animals were examined for blood chemistry and histology of gut
tissues. Controls were treated in the same way, but received salt
water instead of oil.
The same five series of experiments were performed on sockeye salmon.
In all series, statistical significance was determined from 2x2 con-
tingency tables (1, 3).
PHASE II
This phase of the work involved testing various crude oil components
for toxicity. For reasons given in the discussion section, environ-
mental parameters were not monitored, tissues were not taken, and only
those blood parameters were examined which had appeared significant
in earlier experiments. All experiments were conducted with coho
salmon. Average weight of fish ranged from about 5 grams each in the
fall of the year to nearly 40 grams in May. The number of fish per tank
was adjusted to provide a ratio of 1 gram or less of fish per liter
of water.
After the fish had been acclimated, as in Phase I, various amounts of
test substance were introduced into the tanks, simply by squirting
measured volumes from a small syringe. Basic quantities of substance
were the equivalents of 1, 10, and 100 parts per million, but amounts
varied as occasion demanded. Thus, 100 ppm xylene killed all the fish
so quickly that no analyses could be made. Hence, for blood analyses
after exposure to xylene, a concentration of 30 ppm was used.
The following substances were tested:
1. Pentane
2. Hexane
3. Heptane
4. Octane
5. 2-hexene
6. Cyclopentane
7. Ethylcyclopentane
8. Cyclohexane
9. Ethylcyclohexane
10. Cyclopentene
11. Cyclohexene
12. 1,3 cyclohexadiene
13. Benzene
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14. Ethylbenzene
15. Xylene (standard laboratory mixture of isomers)
16. Toluene
As noted in the discussion of Phase I (see page 9), early analyses of
blood chemistry produced inconclusive results. It was not until the
experiment of 8-12 November 1971 that we realized that we had been
analyzing survivors whose blood had returned more or less to normal.
Hence, in Phase II, blood analyses were generally done every hour up
to 4 hours and at 24 hours after exposure to the test substances.
Because of the small size of the fish, it was usually impossible to
obtain enough blood from a single specimen to analyze for all three
ions (Na+, K+, find Cl~), so tests were usually run for sodium and
potassium or for sodium and chloride. Analyses were confined to
those experiments in which significant mortalities of behavioral ab-
normalities were observed, specifically, benzene, toluene, xylene,
and cyclohexene. The experiment with 1,3 cyclohexadiene was limited
to observations of mortality and behavior.
8
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SECTION V
RESULTS
PHASE I
Environmental Parameters
Salinity varied, apparently more or less at random, by about ±0.1 °/oo,
probably because of splatter and evaporation. pH remained constant
at 8.1. Oxygen saturation ranged between 65% and 80%. There was no
detectible free C02. Heavy metals, H2S, and NHj did not vary from the
traces found in the original tap water used to make the artificial sea
water. In view of the constancy of these parameters, they were not
monitored in Phase II.
Blood Parameters
Analyses of blood ions and pH made during Phase I produced only incon-
clusive results. There were indications of rises in Na+, K+, and Cl~,
and a suggestion of a slight decline in pH, In the final experiment
of this phase, analyses were made at 24-hour intervals instead of at
the end of the 96-hour period. These showed a rise in chloride levels
during the first.24 hours, followed by a gradual drop back to normal
levels in the next 2 or 3 days (Table 1). This discovery indicated a
change in approach to more frequent analyses, which was put into effect
in Phase II.
Apparently significant changes in some blood serum proteins were found
in sockeye salmon. Sturdevant (9) reported that the bands designated
by him as 9 and 17 tended to increase in density after exposure to
crude oil.
Tissue Studies
More than 2,000 slides of the previously listed tissues were examined.
No abnormalities were discovered that could definitely be ascribed to
the experimental conditions. Clubbing of gill filaments was the most
common anomaly, but this condition may be caused by any of.several fac-
tors, such as accumulation of nitrogenous compounds in the water (J.
Wallis, Superintendent, Fire Lake Hatchery, pers. comm.), myxobacterial
infection (2), or insufficient pantothenic acid in the diet (5). It
may be that metabolic wastes accumulated under the oil film, that the
action of myxobacteria already present on the gills was enhanced by the
experimental conditions, or that starvation of the fish during the
course of the experiment resulted in a dietary deficiency. None of
these seems probable, and, as some clubbing was present in the controls,
we are without an explanation.
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Table 1. CHANGES IN SERUM CHLORIDE OF YOUNG SOCKEYE SALMON EXPOSED
e
TO VARIOUS AMOUNTS OF CRUDE OIL AT 8 C
Length of ex- Av. Cl~ Std.
posure, hrs mEq/1 dev. n
'3500 ppm oil
24 142.85 18.9865 6
48 150.16 19.1951 6
72 124.17 - 2
96 125.08 - 3
1750 ppm oil
24 162.43 22.6596 5
48 147.68 18.0647 5
72 140.27 16.2246 9
96 - - 0
500 ppm oil
24 - - 0
48 150.82 16.4617 8
72 138.80 18.1560 5
96 126.85 10.6888 6
Control
126.95 8.7983 29
10
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A few slides of stomach tissue from fish that had been force-fed with
crude oil seemed to have thinner mucosa layers than did controls.
No other tissue abnormalities were noted.
Mortalities
Series I
Coho salmon-Experiment I-C-1, begun on 4 December 1970, involved
exposing fish to 3500 ppm oil at each of three different temperatures.
Mortalities after 72 hours are shown in Table 2. During the following
24 hours, the temperature controls on the 3° tanks failed, apparently
because of leakage of refrigerant. Temperatures in these tanks varied
during the night by as much as 7° C, and all fish in them died. For
the remaining tanks, 96 hour mortalities are listed in Table 3.
Experiment I-C-2 began on 4 January 1971. It was the same as the pre-
vious experiment, but without the 3° C tanks. The oil used was a
second aliquot of the original crude oil sample, and had been kept
in a sealed five-gallon can, with considerable air space above the
surface of the oil, for a month. After 96 hours, 12.5% of the 13° C
experimental and 12% of the 8° C experimental had died, compared
with 10% of the 13° C controls and none of the 8° C controls. The
experimental death rate was not significantly different from that of
the controls (Table 4).
A new sample of oil was obtained and experiment I-C-3 was begun on
12 February 1971. This experiment used 3500 ppm of oil at 8° C.
Results are listed in Table 5.
Sockeye salmon-Sockeye salmon became available in Late June 1971.
Experiment I-S-1 was begun on 8 July 1971. All tanks were held at
8° C. The various amounts of oil per tank and the resulting mortal-
ities are shown in Table 6.
Experiment I-S-2, begun on 30 July 1971, was a repetition of I-S-1,
but at temperatures of 3°-5° C. Some of the tanks would still not
hold these low temperatures, hence the number of tanks involved was
less than in previous experiments. Results are shown in Table 7.
Experiment I-S-3, begun on 12 August 1971, was again a repetition of
I-S-1, but with all tanks at 13° C. Results are listed in Table 8.
Series II
Coho salmon-In experiment II-C-1, conditions were the same as in
Series I, except that in half the tanks the oil was physically mixed
with the water. Mortalities are given in Table 9.
11
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Table 2. 72 HOUR MORTALITIES IN YOUNG COHO SALMON EXPOSED TO CRUDE
OIL IN EXPERIMENT I-C-1
Tank
3
7
8
4
5
17
1
2
9
11
13
10
12
14
15
18
Temp.
°C
3
3
3
8
8
8
8
8
8
8
8
13
13
13
13
13
Oil Cone.
ppm equiv.
control
3500
3500
control
control
control
3500
3500
3500
3500
3500
control
3500
3500
3500
3500
72 hour
mortality
0 of 10
2 of 10
7 of 10
1 of 10
0 of 10
2 of 10
7 of 10
9 of 10
0 of 10
0 of 10
5 of 10
0 of 10
8 of 10
3 of 10
1 of 10
8 of 10
Average
mortality
0%
45%
10%
42%
0%
50%
P
<0.05
<0.02
0.02
12
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Table 3. 96 HOUR MORTALITIES IN YOUNG GOHO SALMON EXPOSED TO CRUDE
OIL IN EXPERIMENT I-C-1
Tank
Temp
Oil cone.
ppm equiv.
96 hour
mortality
Average
mortality
4
5
17
1
2
9
11
13
10
12
14
15
18
8
8
8
8
8
8
8
8
13
13
13
13
13
control
control
control
350.0
3500
3500
3500
3500
control
3500
3500
3500
3500
of 10
of 10
1
0
1 of 10
10 of 10
10 of 10
1 of 10
of 10
of
0
7
10
0 of 10
9 of 10
3 of 10
3 of 10
10 of 10
10%
56% <0.02
0%
62.5% <0.02
13
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Table 4. MORTALITIES OBSERVED IN YOUNG COHO SALMON EXPOSED TO
3500 PPM OF WEATHERED CRUDE OIL IN EXPERIMENT I-C-2
Temp. Number Number and % dead after
°C of fish 24 hrs 48 hrs 72 hrs 96 hrs
Exptls.
8 50 1 (2%) 4 (8%) 6 (12%) 6 (12%)
13 40 4 (10%) 4 (10%) 5 (12.5%) 5 (12.5%)
Controls
8 10 0 0 0 0
13 10 00 1 (10%) 1 (10%)
14
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Table 5. 96 HOUR MORTALITIES IN YOUNG COHO SALMON EXPOSED TO CRUDE
OIL AT 8° C IN EXPERIMENT I-C-3
Oil Cone., 96 hour Average
Tank ppm mortality mortality P
2 control 1 of 11 9.1%
7 3500 7 of 7
8 3500 8 of 8
17 3500 2 of 6
18 3500 7 of 9
4 3500 9 of 11 80.5% .£0.02
15
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Table 6. 96 HOUR MORTALITIES IN YOUNG SOCKEYE SALMON EXPOSED TO
DIFFERENT AMOUNTS OF CRUDE OIL AT 8° C IN EXPERIMENT I-S-1
Tank
7
8
1
2
18
9
10
11
12
14
15
4
16
17
Oil Cone.,
ppm
control
control
500
500
500
1000
1000
1000
1750
1750
1750
3500
3500
3500
96 hour
mortality
1 of 10
1 of 10
6 of 9
5 of 9
0 of 10
10 of 10
0 of 9
3 of 10
0 of 10
1 of 10
1 of 10
1 of 10
10 of 10
2 of 10
Average
mortality P
10%
39.3% >0.05, 0.10
40% <0.05
16
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Table 7. 96 HOUR MORTALITIES IN YOUNG SOCKEYE SALMON EXPOSED TO
DIFFERENT AMOUNTS OF CRUDE OIL AT 3°-5° C IN EXPERIMENT I-S-2
Oil Cone., 96 hour Average
Tank ppm mortality mortality P
17 control 0 of 10
18 control 0 of 10 0%
1 500 7 of 10
2 500 4 of 10 55% <0.01
11 1000 10 of 10
12 1000 10 of 10 100% <0.01
7 1750 10 of 10
8 1750 10 of 10 100% <0.01
14 3500 10 of 10
15 3500 8 of 10 90% <0.01
17
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Table 8. 96 HOUR MORTALITIES IN YOUNG SOCKEYE SALMON EXPOSED TO
DIFFERENT AMOUNTS OF CRUDE OIL AT 13° C IN EXPERIMENT I-S-3
Oil Cone., 96 hour Average
Tank ppm mortality mortality P
1 control 0 of 10
4 control 0 of 10
8 control 0 of 10
9 control 0 of 10 0%
11 500 0 of 10
13 500 0 of 10 0% >0.10
7 1000 1 of 9
14 1000 0 of 10
15 1000 1 of 10 6.9% >0.10
16 1750 0 of 10
17 1750 1 of 10 5% >0.10
2 3500 5 of 10
5 3500 1 of 10
18 3500 0 of 10 20% 0.02
18
-------
Table 9. 96 HOUR MORTALITIES IN YOUNG COHO SALMON EXPOSED TO MIXED
OR UNMIXED CRUDE OIL AT 8° C IN EXPERIMENT II-C-1
Unmixed Series
Mixed Series
Oil Cone.,
ppm
0
10
100
300
625
1250
2500
0
10
100
300
625
1250
2500
Number of
fish
5
5
5
5
5
5
5
5
5
5
5
5
5
5
Number and %
dead
0
0
4 (80%)
3 (60%)
0
0
1 (20%)
0
0
0
0
0
0
3 T60%)
19
-------
Series III and IV
The results of these experiments are listed in Table 10. Although
there was some mortality among the experimentals, it was not sta-
tistically significant.
Series V
In the ingestion experiments of Series V, the mortalities in the ex-
perimental series did not differ significantly from those of controls.
Behavior
The young salmon of both species, when subjected to direct exposure to
crude oil (Series I and II) began to show signs of stress, or at least
abnormal behavior, in as little as 45 minutes after the oil had been
introduced into the tanks. The first indication was that all fish
under oil slicks of 500 ppm equivalent or greater began to swim at the
surface of the water with their dorsal fins and the upper lobes of
the caudal fins in the oil film.
A second indication of stress was a loss of equilibrium. This generally
began to appear within 24 hours. The salmon tilted to one side and
their swimming became weak. Subsequently, affected individuals assumed
a head-up, tail-down position, which eventually might become vertical.
All individuals did not show these symptoms. Of those that reached the
vertical position, only a very few recovered. Salmon that did recover
have been kept under oil films for as long as 30 days. These experi-
ments involved a single application of oil; presumably most of the
vplatiles had disappeared by the time the fish recovered.
Salmon of both species that had been force-fed with oil showed no
abnormal behavior except that, within 2 hours of recovery from anes-
thesia, they began to excrete large amounts of oil and mucus. This
continued for up to 12 hours, by which time it is assumed that all the
oil had passed through the intestinal tract.
Fish in the experiments of Series III and IV showed no noticeable
abnormal behavior after recovery from anesthesia.
PHASE II
Mortalities
The following did not produce significant mortalities when applied in
amounts up to 100 ppm equivalent:
Pentane
Hexane
20
-------
Table 10. MORTALITIES OBSERVED IN EXPERIMENTS III-C-1, IV-C-1,
III-S-1, and IV-S-1
Number alive after hrs
Tank Condition* 0 24 48 72 96
Experiments III-C-1 and IV-C-1
4 Dip 54444
17 Dip 55555
16 Dip 54444
14 Drop 55555
15 Drop 55555
12 Control 66666
Experiments III-S-1 and IV-S-1
1 13° Dip 10 10 8 8 7
2 13° Drop 10 10 9 9 9
4 13° Control 11 11 11 11 11
15 8° Dip 10 10 10 10 9
16 8° Drop 10 10 9 9 9
17 8° Control 10 10 10 10 10
*Dip: fish dipped into a thick film of oil, then placed in a tank
of clean water.
Drop: one drop of crude oil placed directly on first gill of each
side, fish then placed in tank of clean water.
21
-------
Heptane
Octane
Cyclopentane
Ethylcyclopentane
Cyclohexane
Ethylcyclohexane
Cyclopentene
Cyclohexene
Significant mortalities were produced with all of the following:
1,3 cyclohexadiene
Benzene
Ethylbenzene
Xylene
Toluene
The results for the last five compounds are listed in Tables 11
through 15.
Behavior
The behavior of fish treated with the various pure substances was,
in general, like a speeded-up and exaggerated version of the behavior
under crude oil.
Pentane, hexane, heptane, and octane produced the least pronounced
reactions. No abnormalities were observed at low concentrations,
but when 100 ppm of these substances were applied, the fish showed
signs of mild irritation. These consisted of rather rapid, erratic
movement, and "coughing." In the latter, the fish opened their mouths
and "backed water" with their pectoral fins, meanwhile holding their
bodies straight. When 500 and 1000 ppm were used, the same symptoms
were more pronounced and persisted for up to 72 hours.
No noticeable alterations of behavior were found in the experiments
with 2-hexene, cyclopentane, ethylcyclopentane, cyclohexane, and
ethylcyclohexane.
With cyclopentene, the increased activity and "coughing" appeared within
10 minutes of application of 50 and 100 ppm equivalents. These pheno-
mena were much reduced after 2 hours and had disappeared by 4 hours
after initial exposure.
The reactions of the fish to 100 ppm of cyclohexene were very different
from the reactions to any other substance tested. There was no sign
of erratic swimming or of "coughing." Instead, within 30 minutes of
application of the cyclohexene, the fish began to undergo "spasms."
All the fins were fully spread, the body appeared to become rigid, and
the fish quivered for several seconds. Upon relaxation, the fish swam
normally for several minutes, then "spasmed" again. This went on for
3 to 4 hours, after which the fish once again seemed entirely normal.
22
-------
Table 11. MORTALITIES PRODUCED WITH YOUNG COHO SALMON AND 50 PPM OF
1, 3 CYCLOHEXADIENE AT 8° C
Tank
5 control
6 control
9 control
17
1
2
4
8
7
11
10
12
18
0
3
3
3
3
3
3
3
3
3
3
3
3
3
Number
24
3
3
3
2
2
2
3
3
2
1
1
2
2
alive
48
3
3
3
2
2
2
3
3
2
1
1
2
2
after
72
3
3
3
2
2
2
3
3
2
1
1
2
2
hrs %
96 mortality
3
3
3 0
2
2
2
3
3
2
1
1
2
2 33.3
P
<0.005
23
-------
Table
AT 8°
Tank
16
17
L8
12
14
15
1
2
4
10
11
7
8
9
12. MORTALITIES PRODUCED WITH
C
Cone.
ppm
control
control
control
1
1
1
10
10
10
50
50
100
100
100
YOUNG COHO SALMON AND
BENZENE
Number alive after hrs %
0 24 48 72 96 mortality P
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
9
10
10
10
10
10
3
6
0
0
0
10
10
10
9
10
10
10
10
10
3
5
0
0
0
9
10
10
9
10
10
10
10
10
3
5
0
0
0
9
10
10 3.3
9
10
10 3.3
M
9
10 3.3
3
5 60
0
0
0 100
>0.05
>0.05
<0.005
<0.005
24
-------
Table 13. MORTALITIES PRODUCED WITH YOUNG COHO SALMON AND ETHYL-
BENZENE AT 8° C
Tank
18
13
16
15
14
12
11
10
8
Cone.
ppm
control
control
control
10
10
10
50
50
50
Number alive after hrs
0 24 48 72 96
10
10
10
10
10
10
10
10
10
10
9
10
9
9
10
0
0
0
9
9
10
9
9
10
0
0
0
9
9
10
8
9
10
0
0
0
9
9
10
8
8
10
0
f 0
0
%
mortality P
6.7
13.3 >0.05
100 < 0.005
25
-------
Table 14. MORTALITIES PRODUCED WITH YOUNG COHO SALMON AND XYLENE
AT 8° C
Tank
8
14
18
4
7
15
9
12
16
TO
11
17
Cone.
ppm
control
control
control
1
1
1
10
10
10
100
100
100
Number alive after hrs.
0 24 48 72
10
10
10
10
10
10
10
10
10
10
10
10
9
10
10
10
10
9
a
9
10
0
0
0
9
9
8
9
10
7
7
8
9
0
0
0
9
9
7
9
9
6
7
8
7
0
0
0
96
8
8
7
8
9
6
7
7
7
0
0
0
%
mortality P
. 23.3
23.3 >0.05
30 >0.05
100 <0.005
26
-------
Table
AT 8°
15. MORTALITIES PRODUCED WITH YOUNG COHO SALMON AND TOLUENE
C
Tank
16
17
18
12
14
15
8
9
11
7
1
2
4
Cone.
ppm
control
control
control
1
1
1
10
10
10
50
100
100
100
Number alive after hrs
0 24 48 72
10
10
10
10
10
10
10
10
10
10
10
10
10
9
10
9
10
9
9
10
10
10
1
1
0
1
9
10
9
10
9
9
10
10
10
0
0
0
0
7
10
9
9
9
8
9
10
10
0
0
0
0
%
96 mortality P
7
10
9
9
9
8
9
8
10
0
0
0
0
13.3
13.3 >0.05
10 >0.05
100 <0.005
100 <0.005
27
-------
At 50 ppm, also, a few fish "spasmed," but the reactions were not so
severe and disappeared in less than 2 hours.
The remaining test substances, 1,3 cyclohexadiene, benzene, ethyl-
benzene, xylene, and toluene, at 20 to 100 ppm, all produced what
appeared to be a single set of behavioral abnormalities. These began,
within 15 to 20 minutes of application, with rapid, violent, and erratic
.swimming. The fish dashed about the aquaria, leaping out of the water,
banging against the sides and covers of the tanks. This was followed
by "coughing," loss of equilibrium, and death. Fish that survived the
first few hours of exposure generally survived to the end of the
experiment (see Tables 11-15).
Blood Chemistry
Results of the blood analyses are listed in tables 16-20. In almost
every analysis, there is a distinct initial rise in the concentration
of the ion concerned, followed by a decline to about the level ob-
served in the controls. This pattern is most obvious with sodium,
less so with potassium and chloride.
28
-------
Table 16. CHANGES IN BLOOD SODIUM AND CHLORIDE ION OF YOUNG COHO
SALMON AFTER EXPOSURE TO 100 PPM CYCLOHEXENE AT 8° C
Time
control
1 hour
2 hours
3 hours
control
1 hour
2 hours
3 hours
X
raEq/1
Sodium
175.19
194.33
173.25
170.25
Chloride
133.94
164.00
153.89
131.14
s
11.8912
31.0648
15.3357
16.4094
6.9806
8; 5599
5.1735
6.8529
n
18
20
20
20
16
12
19
14
-------
Table 17. CHANGES IN BLOOD SODIUM AND POTASSIUM OF YOUNG COHO SALMON
AFTER EXPOSURE TO 15 PPM BENZENE AT 8° C
Time
control
1 hour
2 hours
3 hours
4 hours
24 hours
control
1 hour
2 hours
3 hours
4 hours
24 hours
X
mEq/1
133.81
160.24
157.36
160.33
189.58
153.68
4.34
4.81
4.70
5.02
4.01
3.87
s
Sodium
5.4920
24.8600
7.6405
17.8384
24.6917
29.8161
Potassium
0.3543
0.5040
0. 8788
0.8312
0.3708
0.4451
n
16
16
16
16
14+2 off top of scale
14
16
16
16
16
16
14
30
-------
Table 18. CHANGES IN BLOOD SODIUM AND POTASSIUM OF YOUNG COHO SALMON
AFTER EXPOSURE TO 30 PPM XYLENE AT 8° C
Time
x
mEq/1
n
control
1 hour
2 hours
3 hours
4 hours
24 hours
control
1 hour
2 hours
3 hours
4 hours
24 hours
229.17
233.22
212.98
200.11
248.34
177.09
4.43
4.57
3.70
4.62
4.84
3.96
Sodium
32.9875
14.2992
22.6155
11.2735
17.1728
16.4858
Potassium
0.9013
0.5167
0.6631
0.9524
1.0422
1.3707
14+2 off top of scale
16
14+2 clotted
16
8
16
16
16
16
16
8
16
31
-------
Table 19. CHANGES IN BLOOD SODIUM AND CHLORIDE OF YOUNG COHO SALMON
AFTER EXPOSURE TO 30 PPM XYLENE AT 8° C
Time mEq/1 s n
Sodium
control 186.39 18.5469 18
1 hour 248.86 13.2331 18
3 hours 210.08 16.8551 20
5 hours 197.25 24.7972 20
Chloride
control 134.47 9.2398 19
1 hour 134.75 9.9028 16
3 hours 137.10 10.3004 10
5 hours 140.06 5.6858 16
32
-------
Table 20. CHANGES IN BLOOD SODIUM AND POTASSIUM OF YOUNG COHO SALMON
AFTER EXPOSURE TO 30 PPM TOLUENE AT 8° C
Time
x
mEq/1
n
control
1 hour
2 hours
3 hours
4 hours
control
1 hour
2 hours
3 hours
4 hours
208.19
624.60
225.84
215.84
220.01
4.06
4.75
5.83
5.11
4.89
Sodium
37.6502
11.3566
26.4360
47.9977
44.3942
Potassium
0.7699
1.0571
0.5223
0.9596
0.8110
12+2 off top of scale
4+12 off top of scale
8+2 off top of scale
10+6 off top of scale
8+8 off top of scale
16
16
10
14
16
33
-------
SECTION VI
DISCUSSION
Sturdevant's (9) results with serum proteins appear to be clear-cut.
Bands 9 and 17 on the electrophoresis gel both increased in density
after exposure to crude oil. Band 9 lay in a position corresponding
to the hemoglobin region of human serum, while band 17 lay in the
albumin region. Despite a rather high degree of variation in the data,
which he ascribed to various factors such as individual variation in
the test animals, age and condition of reagents, and operator incon-
sistence, Sturdevant was reasonably certain of the following:
1. Serum proteins of sockeye salmon responded to exposure of
the animal to crude oil.
2. Length of exposure was significant in the effects produced
on both bands.
3. Increased concentrations of oil produced increased effects on
band 9 but not on band 17.
Sturdevant did not attempt to assess the physiological value of these
effects, as he was unable to identify the proteins involved.
The mortalities observed in Phase I, wherein fish were exposed to
Prudhoe Bay .crude oil, show that this oil is highly toxic. Quantities
of 500 ppm equivalent, or greater, produced mortalities that were
statistically significant when compared to mortalities of controls.
This concentration could easily occur if spilled oil were swept into
a shallow cove, for example. Whether or not free-living fish could
and would escape from underneath such a slick is unknown.
The lack of significant mortality in experiment I-C-2 is of particular
interest. The oil used in this experiment had been exposed for 30
days to the large volume of air in the container. It was remarked when
the can was re-opened that the oil was much less odorous than it had
been a month earlier. It is reasonable to assume that during this
period most of the volatile toxic materials in the oil had evaporated,
resulting in the negligible death rates of this experiment.
The experiments with sockeye salmon suggest that, at least for this
species, deaths are inversely related to temperature. Several explan-
ations are possible. One is that at the low temperatures (3°-5° C),
the volatile substances evaporate more slowly, hence the fish are
exposed to higher concentrations for a longer time. It is also pos-
sible that the fish are physiologically less able at low temperatures
and hence succumb more readily to adverse conditions.
In experiment II-C-1, oil was poured in the surface of the water in half
the experimental tanks, but was physically mixed with the water
34
-------
in the remaining experimental tanks. The results of this experiment
are peculiar. The mortalities in individual tanks wherein deaths
occurred are significant, as compared with the controls. However, the
overall mortality in all experimental tanks is not significant, and
we have no explanation.
On the basis of our experimental results, the aromatic hydrocarbon
toxicity increases in the following order: cyclohexane, cyclohexene,
1, 3 cyclohexadiene, toluene, xylene, benzene, and ethylbenzene.
The first part of the series clearly correlates toxicity with the
number of unsaturated bonds.
At the higher concentrations, most deaths occurred within the first
few hours. However, at lower concentrations, fish that survived these
first hours of exposure generally survived to the end of the experiment.
It seems fairly obvious that survival in the experiments of Phase II
was related to the volatility of the test substance and its structure.
A poly-unsaturated, six-carbon-atom ring seemed to be the toxic agent.
The unsaturated cyclic hydrocarbons were the most toxic materials
tested and were also the most volatile. A fish that could withstand
the first few hours, when the concentration of test substance was
highest, stood a good chance of surviving the experiment, for the
volatile nature of the test substances resulted in rapidly decreasing
concentrations as time went on.
The blood analyses done in Phase II generally show a rise in concen-
tration of monovalent ions during the first few hours after exposure,
followed by a return towards the control level. The mean values found
in the experimental fish often are not significantly different from
the control values. Nevertheless, the pattern is too consistent to
be ignored. Likewise, few of the experimental values fall outside the
ranges given by Holmes and Donaldson (4) for 0_. kisutch and related
species. It is quite possible that the rapid, uncontrolled change in
ion concentration, rather than the concentration per se, is the
important factor.
The mechanism of toxicity is still not entirely clear, but the
evidence leads to some reasonable speculations.
The behavior of the fish in response to oil and to pure substances
is, in general, typical of the response of fishes to toxic hydrocarbons
(C. Bond, Department of Fisheries and Wildlife, Oregon State University,
pers. coram.). Swimming at the surface, with the dorsal and caudal
fins touching the oil slick, is difficult to interpret. It is not a
response to reduced light, for fish in tanks covered with heavy black
paper retained the usual, more or less random distribution, while those
in tanks covered with oil, but brightly illuminated from the side,
swam at the surface. Likewise, it is not a reaction to insufficient
35
-------
oxygen, for full aeration was maintained and dissolved oxygen was
always between 65% and 80% saturation. However, other aspects of
the observed behavior are more amenable to interpretation.
The fact that the most toxic materials tested are fat solvents sug-
gests that the permeability of membranes, particularly in the gills, is
increased through the loss of fatty substances. This idea is sup-
ported by the observed increases in the concentrations of the mono-
valent ions sodium, potassium, and chloride in the blood. Since our
fish were tested in a hypertonic environment, increased intake of
monovalent ions would be expected if the permeability of the gill
membranes were increased. (Parenthetically, the ability of the fish
to restore the fat balance in these tissues must be quite rapid. At
any rate, blood ions approached normal concentrations almost as
rapidly as the volatile test materials were lost from the tanks.)
The increased ion concentrations in the blood probably interfere with
carbonate and pH adjustment. It is reasonable to assume that the
chloride shift has been altered. This, in turn, would affect the
CO^-HCOg balance and could interfere with the fish's ability to control
the gas content of the swim bladder. Loss of this control could
account for the loss of equilibrium. Loss of equilibrium, particularly
the head-up, tail-down position, accompanied by weakened swimming
movements, is a typical symptom of C02 poisoning (10). If the fish
cannot control the C02-HC03 balance in its blood, internal accumu-
lation of C02 may be a contributing factor in producing death.
Very little is known of the symptoms of ionic imbalance in fishes,
but by analogy with mammalian and especially human symptoms, it seems
probable that this, too, is an important factor. In particular,
muscular hypertension, which was noted in several experiments, and
loss of muscular control, may be attributed to ionic imbalance.
In summary, then, the.toxicity of crude oil to fishes is most likely
attributable to unsaturated cyclic compounds in the oil. These
compounds probably act by increasing the cell membrane permeability
of the gills, resulting in ionic imbalance and internal C02 poisoning.
36
-------
SECTION VII
REFERENCES
1. Bennet, B. M., and C. Horst. Supplement to Tables for Testing
Significance in a 2x2 Contingency Table. Cambridge, England,
Cambridge U. Press, 1966. 28 p.
2. Davis, H. S. Culture and Diseases of Game Fishes. Berkeley and
Los Angeles, U. of California Press, 1961. p. 261-264; 281-282.
3. Finney, D. J., R. Latscha, B. M. Bennett, and P. Hsu. Tables for
Testing Significance in a 2x2 Contingency Table. Cambridge,
England, Cambridge U. Press, 1963. 103 p.
4. Holmes, W. N., and E. M. Donaldson. The Body Compartments and the
Distribution of Electrolytes. In: Fish Physiology, Vol. 1, W. S.
Hoar and D. J. Randall (eds.). New York and London, Academic
Press, 1969. p. 1-89.
5. Rucker, R. R., ,H. E. Johnson, and G. M. Kaydas. An Interim Report
on Gill Disease. Prog. Fish-Cult. J14_(1) :10-14, January 1952.
6. Schales, 0., and S. S. Schales. A Simple and Accurate Method for
the Determination of Chloride in Biological Fluids. J. Biol.
Chem. 14CI(3): 879-884, 1941.
7. Scripps Institution of Oceanography. Oceanic Observations of the
Pacific: 1955. Berkeley and Los Angeles, U. of California Press,
1962.
8. Strickland, J. D. H., and T. R. Parsons. A Practical Handbook of
Seawater Analysis. Bull. Fish. Res~ Bd. Canada, Ottawa 167:21-53,
1968.
9. Sturdevant, D. C. The Influence of Crude Oil on the Serum Proteins
of the Sockeye Salmon. Fairbanks, M.S. Thesis, U. of Alaska,
May, 1972. 43 -p.
10. van Duijn, C., Jr. Diseases of Fishes. 2 ed. Springfield, Charles
C Thomas, 1967. p. 93-95.
37
-------
1
Accession Number
5
2
5ub;'ecf Field & Group
SELECTED WATER RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
Organization
DeuartmeTiti of Biological Sciences
University of Alaska
Title
Effects of Crude Oil and Some of its Components on Young Coho and Sockeye Salmon
10
Authors)
James E. Morrow
16
Project Designation
R 801039 (formerly 16100FWQ)
2] Note
22
citation
Protection Agency report
number EPA-660/3-73-018, January 1974.
23
Descriptors (Started First)
*0il pollution; *marine environment; Prince William Sound;
25
Identifiers (Starred First)
*Crude oil; *Coho salmon; *Sockeye salmon; aliphatic hydrocarbons; aromatic
hydrocarbons; monovalent blood ions.
27
Abstract
Young coho and sockeye salmon, acclimated to 30 /oo salinity, were exposed in
various ways to different amounts of crude oil from the Prudhoe Bay field. Oil
poured on the surface of the water in 95 liter (25 gallon) aquaria produced sig-
nificant mortalities when the oil concentration was 500 ppm or greater. Fish
dipped into a crude oil film, or with a drop of oil placed directly on each gill,
showed no significant mortalities. The same was true of fish force-fed crude oil
at 1 g per 100 g body weight. Oil that had been exposed to air for 30 days pro-
duced no significant mortalities.
Among oil components tested for toxicity on coho salmon, aliphatic compounds
were not lethal. Mono-cyclic aromatics were generally toxic, the degree of
toxicity increasing with the degree of unsaturation.
It is suggested that the toxicity of these substances is brought about through
alteration of cell membrane permeability, especially in the gills. This results
in a rapid increase of mono-valent ions in the blood and probably also interferes
with CO -HCO" regulation.
Abstractor
J. E. Morrow
Institution
University of Alaska
WR:102 (REV. JULY 1969)
WRSIC
SEND TO: WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON. D- C. 20240
GPO: 1969-359-339
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